Manufacture of peanut formulations for oral desensitization

ABSTRACT

The present application relates to a method for managing the development and manufacturing process of a therapeutically effective formulation. Peanut proteins are characterized from peanut flour and encapsulated formulations made using the peanut flour for oral immunotherapy of peanut allergies.

CROSS REFERENCE

This application claims the benefit of U.S. Provisional Application No.61/784,964, filed Mar. 14, 2013, which application is incorporatedherein by reference in its entirety.

This application is related to U.S. Provisional Application No.61/784,863, filed Mar. 14, 2013, entitled “Peanut Formulations and UsesThereof” (Attorney Docket No. 43567-702.101), which is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

Allergies affect humans and companion animals and some allergicreactions (for example, those to insects, foods, latex, and drugs) canbe so severe as to be life threatening.

Allergic reactions result when a subject's immune system responds to anallergen. Typically, there is no allergic reaction the first time asubject is exposed to a particular allergen. However, it is the initialresponse to an allergen that primes the system for subsequent allergicreactions. In particular, the allergen is taken up by antigen presentingcells (APCs; e.g., macrophages and dendritic cells) that degrade theallergen and then display allergen fragments to T-cells. T-cells, inparticular CD4+ “helper” T-cells, respond by secreting a collection ofcytokines that have effects on other immune system cells. The profile ofcytokines secreted by responding CD4+ T-cells determines whethersubsequent exposures to the allergen will induce allergic reactions. Twoclasses of CD4+ T-cells (Th1 and Th2; T-lymphocyte helper type)influence the type of immune response that is mounted against anallergen.

The Th1-type immune response involves the stimulation of cellularimmunity to allergens and infectious agents and is characterized by thesecretion of IL-2, IL-6, IL-12, IFN-gamma, and TNF-beta by CD4+ T helpercells and the production of IgG antibodies. Exposure of CD4+ T-cells toallergens can also activate the cells to develop into Th2 cells, whichsecrete IL-4, IL-5, IL-10, and IL-13. IL-4 production stimulatesmaturation of B cells that produce IgE antibodies specific for theallergen. These allergen-specific IgE antibodies attach mast cell andbasophil receptors, where they initiate a rapid immune response to thenext exposure to allergen. When the subject encounters the allergen asecond time, the allergen is quickly bound by these surface-associatedIgE molecules, resulting in the release of histamines and othersubstances that trigger allergic reactions. Subjects with high levels ofIgE antibodies are known to be particularly prone to allergies.

SUMMARY OF THE INVENTION

Provided herein is a method of making a low dose capsule formulationuseful in the methods provided here, comprising, (a) mixing peanut flourand diluent in a first blend; (b) adding about 45% of diluent in asecond blend; (c) adding remaining diluent in a third blend; (d) addinga glidant and/or lubricant in a final blend; and (e) encapsulatingblended powder in a capsule. In one embodiment, the diluent of step (a)comprises starch or lactose, microcrystalline cellulose (Avicel®), ordicalcium phosphate. In another embodiment, the diluent of step (b)and/or (c) comprises starch, lactose, microcrystalline cellulose(Avicel®), or dicalcium phosphate. In another embodiment, the glidant ofstep (d) glidant of step (d) comprises colloidal silicon dioxide(Cab-O-Sil), talc (e.g., Ultra Talc 4000), or combinations thereof. Inanother embodiment, the lubricant of step (d) comprises magnesiumstearate. In one non-limiting example, the glidant comprises Cab-O-Sil.In one embodiment, step (d) comprises adding a glidant or a lubricant.In another embodiment, step (d) comprises adding a glidant and alubricant. In another embodiment, the method further comprises samplingthe blended mixture one or more times prior to encapsulation. In anotherembodiment, the dose comprises about 0.5 or about 1.0 mg peanut protein.In another embodiment of the described methods, step (d) furthercomprises passing the blended material through a mesh screen.

Provided herein is a method of making a higher dose capsule formulationuseful in the methods provided here, comprising, (a) mixing peanut flourand diluent in a first blend; (b) discharging the blended material; (c)passing the blended material through a mesh screen and blending thescreened material in a second blend; (d) adding in a glidant and/orlubricant in a third blend; and (e) encapsulating the blended powder. Inone embodiment, the method optionally comprises sampling the blendedmaterial of step (d) one or more times prior to encapsulation. In yetanother embodiment, the diluent of step (a) comprises starch, lactose ormicrocrystalline cellulose (Avicel®), or dicalcium phosphate. In anotherembodiment, the mesh screen of step (c) comprises a #20 mesh screen. Inanother embodiment, the glidant of step (d) glidant of step (d)comprises colloidal silicon dioxide (Cab-O-Sil), talc (e.g., Ultra Talc4000), or combinations thereof. In another embodiment, the glidant ofstep (d) comprises Cab-O-Sil. In another embodiment, the lubricant ofstep (d) comprises magnesium stearate. In one embodiment, step (d)comprises adding a glidant or a lubricant. In another embodiment, step(d) comprises adding a glidant and a lubricant. In another embodiment,the dose comprises about 10, about 100 or about 475 mg peanut protein.

Provided herein is a method of making a capsule formulation useful inthe methods provided here, comprising, passing peanut flour through amesh screen; and encapsulating the blended powder. In one embodiment,the dose comprises about 475 mg peanut protein.

In any of such methods, the peanut flour may comprise characterizedpeanut proteins. In one embodiment, the peanut proteins comprise Ara h1,Ara h2 and Ara h6. The concentration of Ara h1, Ara h2 and Ara h6 may becharacterized by RP-HPLC. In another embodiment, the concentration ofAra h1, Ara h2 and Ara h6 is at least an amount of a reference standard.

An encapsulated formulation produced by any of the methods describedherein may be stable for at least about 3, 6, 9, 12, 18, 24, 36 or moremonths.

In one embodiment, the encapsulated formulation is stable at atemperature from about 2° C. to about 8° C. or from about 20° C. toabout 30° C.

In another embodiment, the encapsulated formulation is stable at atemperature of about 20° C., about 21° C., about 22.5° C., about 23° C.,about 24° C., about 25° C., about 26° C., about 27.5° C., about 28° C.,about 29° C., or about 30° C.

A capsule size that may be used to hold the formulations produced by themethods described herein may be, for example, size 3, 00 or 000. In oneembodiment, the capsule comprises Hydroxypropyl Methyl Cellulose (HPMC).

The methods described herein may further comprise storing a formulationin a container means. Any suitable container means may be used to storethe encapsulated formulations described herein. In one embodiment, thecontainer means may be, for example, a bottle. A bottle may be, forexample, an amber-colored bottle in order to minimize exposure of theencapsulated formulations to ultraviolet light. In another embodiment,the container means further comprises a dessicant packet to controlmoisture content of the container means.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1: Peanut flour extract at 214 nm using reversed phase HPLC. USDAAra h standards, along with a 1 mg/mL BSA solution are also shown. Theextracts are as follows: Top panel: Peanut flour, pH 8.2 extract; secondpanel: Ara h1 peak; third panel: Ara h2 peak; fourth panel: Ara h6 peak;bottom panel: 1 mg/ml BSA solution.

FIG. 2: Chromatograph results from RP-HPLC analysis of 112FA02411 (GMP).

FIG. 3: Chromatograph results from RP-HPLC analysis of 112FA02411 (NonGMP).

FIG. 4: Chromatograph results from RP-HPLC analysis of 111FA36211 (NonGMP).

FIG. 5: Total Protein Staining of Pooled and RP-HPLC Purified Ara hProteins.

FIG. 6: Immunoblots of Pooled and RP-HPLC Purified Ara h Proteins.

FIG. 7: Blending Process Flow Diagram for Low Dose Capsules (0.5 mg and1 mg).

FIG. 8: Blending Process for the High Dose Capsules (at least 10 mg).

FIG. 9: Chromatogram results from RP-HLPC analysis of 112FA02411 (GMP).

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are systems and methods that isolate proteins frompeanut flour, which may be used to manufacture pharmaceuticalcompositions for treatment of peanut allergies. The systems and methodsutilize high pressure (phase) liquid chromatography (HPLC) to captureAra h1, Ara h2 and Ara h6 from peanut flour.

During the past decade, much has been learned about allergens in peanut.Peanuts are commonly associated with severe reactions, including lifethreatening anaphylaxis. The current standard of care in management offood allergy is dietary avoidance of the food and education of thesubject/family in the acute management of an allergic reaction. Theburden of avoidance and constant fear of accidental exposure negativelyimpacts the health-related quality of life for both subjects and theirfamilies. Quality of life surveys indicate that families with childrenhaving food allergies have significant impact on food preparation,social activities, finding appropriate childcare, school attendance, andlevel of stress among other things.

Currently, the only treatment for peanut allergy is a peanut-free dietand ready access to self-injectable epinephrine. However, strictavoidance diets can be complicated due to difficulty in interpretinglabels and by the presence of undeclared or hidden allergens incommercially prepared foods. Accidental ingestions are unfortunatelycommon, with up to 50% of food-allergic subjects having an allergicreaction over a two-year period. Allergic reactions to peanut can besevere and life threatening; and peanut and/or tree nut allergiesaccount for the vast majority of fatal food-induced anaphylaxis. Thiscombination of strict avoidance diets, the high incidence of accidentalexposures, and the risk of severe or even fatal reactions withaccidental exposures adds a tremendous burden and stress on subjects andtheir families. Further complicating matters is the fact that only about20% of children will outgrow peanut allergy, meaning that the majorityof people with peanut allergy will have it for the rest of their lives.If we couple the rising prevalence and increased consumption of peanutin Western countries with the facts that only approximately 1 in 5 willoutgrow their allergy, that allergic reactions have the potential to besevere or even fatal, and that accidental exposures are common,developing an effective treatment for peanut allergy becomes even moreimperative.

Specific immunotherapy for food allergy, in particular peanut allergy,in the forms of oral immunotherapy (OIT) and sublingual immunotherapy(SLIT) has been studied in recent years and has demonstrated encouragingsafety and efficacy results in early clinical trials, includingbeneficial immunologic changes. OIT has shown evidence for inducingdesensitization in most subjects with immunologic changes over timeindicating progression toward clinical tolerance.

Peanut OIT: In Jones et al., peanut allergic children underwent an OITprotocol consisting of an initial dose escalation day, bi-weeklybuild-up (to 2 g) and daily maintenance phase followed by an OFC. Aftertolerating less than 50 mg peanut protein during an oral food challenge(OFC) at baseline, 27 of the 29 subjects ingested 3.9 g of peanutprotein at the completion of OIT protocol.

Recently, Dr. Wesley Burks. (American Academy of Allergy, Asthma, andImmunology National Conference. Orlando, Fla., Mar. 6, 2012) presentedwork showing that 10 children with PA completed an OIT protocol andunderwent an oral food challenge (OFC) 4 weeks after cessation of oralintake of peanut to evaluate the development of clinical “sustainedunresponsiveness”. Three out of 10 subjects passed the OFC; the authorsconsidered these subjects as clinically tolerant. Over the course oftreatment, peanut IgE levels lower than 85 kU/L at a time point of 3months into OIT was predictive of subjects who became immune tolerant.

A multi-center double-blinded randomized placebo-controlled studyreported by Varshney, et al., examined twenty-eight subjects. Threesubjects withdrew early in the study because of allergic side effects.After completing up-dosing, a double-blind placebo-controlled foodchallenge was performed, in which all remaining peanut OIT subjects(n=16) ingested the maximum cumulative dose of 5000 mg (approximately 20peanuts), whereas placebo subjects (n=9) could tolerate only a mediancumulative dose of 280 mg (range, 0-1900 mg; p<0.001). In contrast withthe placebo group, the peanut OIT group showed reductions in skin pricktest size (P<0.001) and increases in peanut-specific IgG4 (P<0.001).Peanut OIT subjects had initial increases in peanut-specific IgE(P<0.01) but did not show significant change from baseline by the timeof oral food challenge.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the inventions described herein belong. All patents andpublications referred to herein are incorporated by reference.

The term “animal”, as used herein, refers to humans as well as non-humananimals, including, for example, mammals, birds, reptiles, amphibians,and fish. Preferably, the non-human animal is a mammal (e.g., a rodent,a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig).An animal may be a transgenic animal.

The term “antigen”, as used herein, refers to a molecule that elicitsproduction of an antibody response (i.e., a humoral response) and/or anantigen-specific reaction with T-cells (i.e., a cellular response) in ananimal.

The term “allergen”, as used herein, refers to a subset of antigenswhich elicit the production of IgE in addition to other isotypes ofantibodies. The terms “allergen”, “natural allergen”, and “wild-typeallergen” may be used interchangeably. Preferred allergens for thepurpose of the present invention are protein allergens.

The phrase “allergic reaction”, as used herein, relates to an immuneresponse that is IgE mediated with clinical symptoms primarily involvingthe cutaneous (e.g., urticana, angiodema, pruritus), respiratory (e.g.,wheezing, coughing, laryngeal edema, rhinorrhea, watery/itching eyes),gastrointestinal (e.g., vomiting, abdominal pain, diarrhea), andcardiovascular (i.e., if a systemic reaction occurs) systems. For thepurposes of the present invention, an asthmatic reaction is consideredto be a form of allergic reaction.

The phrase “anaphylactic allergen”, as used herein, refers to a subsetof allergens that are recognized to present a risk of anaphylacticreaction in allergic individuals when encountered in its natural state,under natural conditions. For example, for the purposes of the presentinvention, pollen allergens, mite allergens, allergens in animal dandersor excretions (e.g., saliva, urine), and fungi allergens are notconsidered to be anaphylactic allergens. On the other hand, foodallergens, insect allergens, and rubber allergens (e.g., from latex) aregenerally considered to be anaphylactic allergens. Food allergens areparticularly preferred anaphylactic allergens for use in the practice ofthe present invention. In particular, legumes (peanuts), tree nutallergens (e.g., from walnut, almond, pecan, cashew, hazelnut,pistachio, pine nut, brazil nut), dairy allergens (e.g., from egg,milk), seed allergens (e.g., from sesame, poppy, mustard), soybean,wheat, and seafood allergens (e.g., from fish, shrimp, crab, lobster,clams, mussels, oysters, scallops, crayfish) are anaphylactic foodallergens according to the present invention. Particularly interestinganaphylactic allergens are those to which reactions are commonly sosevere as to create a risk of death.

The phrase “anaphylaxis” or “anaphylactic reaction”, as used herein,refers to a subset of allergic reactions characterized by mast celldegranulation secondary to cross-linking of the high-affinity IgEreceptor on mast cells and basophils induced by an anaphylactic allergenwith subsequent mediator release and the production of severe systemicpathological responses in target organs, e.g., airway, skin digestivetract, and cardiovascular system. As is known in the art, the severityof an anaphylactic reaction may be monitored, for example, by assayingcutaneous reactions, puffiness around the eyes and mouth, vomiting,and/or diarrhea, followed by respiratory reactions such as wheezing andlabored respiration. The most severe anaphylactic reactions can resultin loss of consciousness and/or death.

The phrase “antigen presenting cell” or “APC”, as used herein, refers tocells which process and present antigens to T-cells to elicit anantigen-specific response, e.g., macrophages and dendritic cells.

When two entities are “associated with” one another as described herein,they are linked by a direct or indirect covalent or non-covalentinteraction. Preferably, the association is covalent. Desirablenon-covalent interactions include, for example, hydrogen bonding, vander Walls interactions, hydrophobic interactions, magnetic interactions,etc.

The phrase “decreased anaphylactic reaction”, as used herein, relates toa decrease in clinical symptoms following treatment of symptomsassociated with exposure to an anaphylactic allergen, which can involveexposure via cutaneous, respiratory, gastrointestinal, and mucosal(e.g., ocular, nasal, and aural) surfaces or a subcutaneous injection(e.g., via a bee sting).

The term “epitope”, as used herein, refers to a binding site includingan amino acid motif of between approximately six and fifteen amino acidswhich can be bound by an immunoglobulin (e.g., IgE, IgG, etc.) orrecognized by a T-cell receptor when presented by an APC in conjunctionwith the major histocompatibility complex (MHC). A linear epitope is onewhere the amino acids are recognized in the context of a simple linearsequence. A conformational epitope is one where the amino acids arerecognized in the context of a particular three dimensional structure.

An allergen “fragment” according to the present invention is any part orportion of the allergen that is smaller than the intact naturalallergen. In preferred embodiments of the invention, the allergen is aprotein and the fragment is a peptide.

The phrase “immunodominant epitope”, as used herein, refers to anepitope which is bound by antibody in a large percentage of thesensitized population or where the titer of the antibody is high,relative to the percentage or titer of antibody reaction to otherepitopes present in the same antigen. In one embodiment, animmunodominant epitope is bound by antibody in more than 50% of thesensitive population, more preferably more than 60%, 70%, 80%, 90%, 95%,or 99%.

The phrase “immunostimulatory sequences” or “ISS”, as used herein,relates to oligodeoxynucleotides of bacterial, viral, or invertebrateorigin that are taken-up by APCs and activate them to express certainmembrane receptors (e.g., B7-1 and B7-2) and secrete various cytokines(e.g., IL-1, IL-6, IL-12, TNF). These oligodeoxynucleotides containunmethylated CpG motifs and when injected into animals in conjunctionwith an antigen, appear to skew the immune response towards a Th1-typeresponse. See, for example, Yamamoto et al., Microbiol. Immunol. 36:983,1992; Krieg et al., Nature 374:546, 1995; Pisetsky, Immunity 5:303,1996; and Zimmerman et al., J. Immunol. 160:3627, 1998.

As used herein, the terms “comprising,” “including,” and “such as” areused in their open, non-limiting sense.

The term “about” is used synonymously with the term “approximately.” Asone of ordinary skill in the art would understand, the exact boundary of“about” will depend on the component of the composition. Illustratively,the use of the term “about” indicates that values slightly outside thecited values, i.e., plus or minus 0.1% to 10%, which are also effectiveand safe. In another embodiment, the use of the term “about” indicatesthat values slightly outside the cited values, i.e., plus or minus 0.1%to 5%, which are also effective and safe. In another embodiment, the useof the term “about” indicates that values slightly outside the citedvalues, i.e., plus or minus 0.1% to 2%, which are also effective andsafe.

“Isolated” (used interchangeably with “substantially pure”) when appliedto polypeptides means a polypeptide or a portion thereof, which has beenseparated from other proteins with which it naturally occurs. Typically,the polypeptide is also substantially (i.e., from at least about 70% toabout 99%) separated from substances such as antibodies or gel matrices(polyacrylamide) which are used to purify it.

Formulations

Formulations described herein include one or more active ingredients.Active ingredients may be isolated from peanut flour which may beobtained from any source such as, for example, the Golden PeanutCompany. The peanut flour may be from about 10% to about 15%, or about12% defatted peanut flour milled from lightly roasted peanuts. Thepeanut flour may be, in some instances, released by a supplier afterstandard analysis of content and microbiology, and may be stable for9-12 months under refrigeration. The peanut flour may be formulated,encapsulated and tested prior to administration to a subject.

For analysis of the peanut flour, bulk substance (BS) and finalformulation, a reverse phase HPLC assay (RP-HPLC) has been developedthat separates three peanut flour protein allergens: Ara h1, Ara h2 andAra h6. This assay forms the basis for identity and content testing atrelease and during stability. The reverse phase-HPLC assay may beutilized as an identification assay and to monitor lot-to-lotconsistency and stability of the peanut allergens acceptable forproduction of the Characterized Peanut Allergen formulation.

Additional characterization of the protein allergens may also beperformed using Enzyme Linked Immunosorbent Assays (ELISA) and gelanalysis.

Peanuts and peanut flour are common foods and additives found in manyfood formulations. The intended clinical use for Characterized PeanutAllergen identified by the present inventors is found in relativelysmall quantities (0.5 to 4000 mg/dose) compared to quantities containedin food and may be delivered via the same route as orally ingestedpeanut-containing products.

Formulations described herein may be tested in a multi-center,placebo-controlled study to demonstrate the safety and efficacy ofCharacterized Peanut Allergen in subjects from about 4 to about 26 yearsof age with moderate-to-severe clinical reactions to peanut ingestion.Subjects with significant concomitant health conditions, uncontrolledasthma, or prior admission to an intensive care unit due to anaphylaxismay be excluded. Standard anti-allergy medications (e.g.,antihistamines, oral corticosteroids, etc.) may be allowed onmaintenance and while up-dosing with Characterized Peanut Allergen(CPA).

A formulation comprising Characterized Peanut Allergen (CPNA), mayinclude peanut protein (comprising peanut allergen proteins Ara h1, Arah2 and Ara h6) formulated with a one or more diluents, one or moreglidants, one or more lubricants and, optionally, one or more fillingagents, in graduated doses, comprising capsules containing about 0.5 mg,about 1 mg, about 10 mg, about 100 mg and about 1000 mg each of peanutprotein. Each capsule may be opened and the content mixed intotaste-masking food immediately prior to administration.

An active pharmaceutical ingredient is initially sourced as raw peanuts,Arachis hypogaea, a member of the legume family. Raw peanuts may beprocured from multiple farming sources, where the shelled, raw peanutsare processed into 12% defatted roasted peanut flour (PF). The PF may becomprise a certificate of analysis (CofA) for further processing undercGMP conditions.

Formulation, fill and testing of the CPNA capsules may be performed at acGMP contract manufacturing site. Under cGMP manufacturing conditions,the protein flour (PF), which is comprised of approximately 50% peanutprotein w/w, is mixed with one or more diluents, one or more glidantsand one or more lubricants.

In one embodiment, a composition comprises one or more diluents.“Diluents” for use in the formulations include, but are not limited to,alginic acid and salts thereof; cellulose derivatives such ascarboxymethylcellulose, methylcellulose (e.g., Methocel®),hydroxypropylmethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose (e.g., Klucel®), ethylcellulose (e.g., Ethocel®),microcrystalline cellulose (e.g., Avicel®); silicified microcrystallinecellulse; microcrystalline dextrose; amylose; magnesium aluminumsilicate; polysaccharide acids; bentonites; gelatin;polyvinylpyrrolidone/vinyl acetate copolymer; crosspovidone; povidone;starch; pregelatinized starch; tragacanth, dextrin, a sugar, such assucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol,xylitol (e.g., Xylitab®), lactose (e.g., lactose monohydrate, lactoseanhydrous, etc.); dicalcium phosphate; a natural or synthetic gum suchas acacia, tragacanth, ghatti gum, mucilage of isapol husks,polyvinylpyrrolidone (e.g., Polyvidone® CL, Kollidon® CL, Polyplasdone®XL-10), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodiumalginate, a starch, e.g., a natural starch such as corn starch or potatostarch, a pregelatinized starch such as Colorcon (Starch 1500), National1551 or Amijel®, or sodium starch glycolate such as Promogel® orExplotab®; a cross-linked starch such as sodium starch glycolate; across-linked polymer such as crospovidone; a cross-linkedpolyvinylpyrrolidone; alginate such as alginic acid or a salt of alginicacid such as sodium alginate; a clay such as Veegum® HV (magnesiumaluminum silicate); a gum such as agar, guar, locust bean, Karaya,pectin, or tragacanth; sodium starch glycolate; bentonite; a naturalsponge; a surfactant; a resin such as a cation-exchange resin; citruspulp; sodium lauryl sulfate; sodium lauryl sulfate in combinationstarch; and combinations thereof. In one embodiment, the formulationcomprises microcrystalline cellulose or starch 1500. In anotherembodiment, the formulation comprises microcrystalline cellulose andstarch 1500.

Suitable glidants (anti-caking agents) for use in the solid dosage formsdescribed herein include, but are not limited to, colloidal silicondioxide (Cab-O-Sil), talc (e.g., Ultra Talc 4000), and combinationsthereof. In one embodiment, the composition comprises Cab-O-Sil.

Suitable lubricants for use in the solid dosage forms described hereininclude, but are not limited to, stearic acid, calcium hydroxide, talc,corn starch, sodium stearyl fumerate, alkali-metal and alkaline earthmetal salts, such as aluminum, calcium, magnesium, zinc, stearic acid,sodium stearates, magnesium stearate, zinc stearate, waxes, Stearowet®,boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, apolyethylene glycol or a methoxypolyethylene glycol such as Carbowax™,PEG 4000, PEG 5000, PEG 6000, propylene glycol, sodium oleate, glycerylbehenate, glyceryl palmitostearate, glyceryl benzoate, magnesium orsodium lauryl sulfate, and combinations thereof. In one embodiment, thecomposition comprises magnesium stearate. In another embodiment, thecomposition comprises sodium stearyl fumerate.

In some embodiments, a formulation may further comprise one or morefilling agents. “Filling agents” include compounds such as lactose,calcium carbonate, calcium phosphate, dibasic calcium phosphate, calciumsulfate, microcrystalline cellulose, cellulose powder, dextrose,dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol,lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, andcombinations thereof.

Ingredients described herein may be mixed according to, for example, theprocesses illustrated in FIGS. 7 and 8. Mixed formulations may besubsequently encapsulated as 0.5, 1, 10, 100 mg, 475 mg, and 1000 mg ofpeanut protein in size 3, 00 or 000. Hydroxypropyl Methyl Cellulose(HPMC) capsules. Compatibility studies may evaluate combinations of thepeanut flour with one or more of the excipients, which may have in someinstances, GRAS recognition. The diluent provides the opportunity toformulate the low and high doses to contain adequate volume fordispersal from the opened capsule. The glidant and lubricant addflowability to the PF such that the capsule is easily emptied of flourby the subject or practitioner at time of administration. For clinicaltrials, the capsules may be bulk packed into a container means such as,for example, bottles. In some instances, the container means may betreated to prevent (partially or fully) exposure to light. For example,a container means may be amber-colored. A container means may also, insome instances, contain a dessicant to prevent (partially or fully)exposure to moisture during shipping and storage. At the time of use,capsule(s) containing CPNA may be opened and the content mixed intotaste-masking food immediately prior to administration.

In order to standardize the delivery of peanut protein allergens, a cGMPmanufactured Characterized Peanut Allergen (CPNA) formulation has beendeveloped. The protein content of the formulation is critical from twoaspects. First, the total protein delivered should be consistent amongbatches, and second, the proportion of critical individual allergensshould be controlled.

Total protein content of the bulk substance and final formulationrelease may be quantified using protein determination methods describedherein which address current issues in the industry: namely, prior tothe present application establishing the absolute or relative amounts ofindividual peanut protein allergens in the peanut flour is moreproblematic and has not been controlled.

Peanut protein is comprised of several individual protein allergenstypically detectable by polyacrylamide gel electrophoresis andimmunoblotting using allergen specific polyclonal antisera from allergichumans or immunized animals. Of these proteins, based on immunoblot,reactivity against crude peanut extracts by human sera from peanutallergic humans, and in vitro histamine release from sensitizedbasophils, Ara h1, Ara h2 and Ara h6 have been identified as allergenicpeanut protein allergens, with Ara h2 and Ara h6 contributing themajority of the allergenic activity of crude peanut extract.

Prior to the present application peanut allergen proteins have typicallybeen fractionated from crude peanut extracts by size exclusionchromatography (SEC) or polyacrylamide gel electrophoresis. Thesetechniques may present a relative view into the spectrum of Araproteins, but do not provide the resolution and sensitivity needed tocompare individual peanut allergen expression among peanut flour lots,nor possible changes in protein structure over time. In order to addressthese limitations, the present inventors have developed a reverse phaseHPLC (RP-HPLC) method to enhance the resolution and allow physicalseparation of peanut allergens Ara h1, Ara h2 and Ara h6.

An assay was developed for the determination and characterization of Arah1, 2 and 6 allergenic proteins in roasted peanut flour. A simple singlestage extraction procedure was modified using Tris buffer at pH 8.2,followed by centrifugation and filtration. Samples are prepared at 100mg/mL and extracted at 60° C. for 3 hours. The final neat filtrate issuitable for direct analysis by HPLC.

The HPLC separation utilizes a reversed phase separation using a widepore 300 Å silica column with a bonded butyl stationary phase. A binarygradient may be employed based upon 0.1% trifluoroacetic acid andacetonitrile. The mobile phase may be compatible with mass spectrometry.Detection may be accomplished with a UV detector at 214 nm, assensitivity may be reduced with detection at 280 nm.

Specificity of the method may be determined by comparing the retentiontimes and peak patterns of the whole peanut extract with Ara h proteins.The principle Ara h protein peaks, in some instances, may not resolve asdiscrete entities, but rather may appear as ensembles of many similarproteins. Thus, the Ara h1, Ara h2 and Ara h6 allergens may appear asclusters of peaks within a retention time region. Accordingly, therelative amount of a particular Ara h protein is then determined as thepercentage of the total area within a defined elution region.Chromatographic resolution of the various regions is assessed, and themethod may be useful for comparison of subtle differences in theseregional patterns for different lots and sources of peanut flourproteins, and stability of the formulation.

A representative example chromatographic series at 214 nm is shown inFIG. 1 comparing the crude extract (top panel) with profiles frompurified Ara h1, Ara h2 and Ara h6 proteins and BSA.

RP-HPLC method pre-qualification may be assessed by comparing threeindependent preparations of a single peanut flour lot, by comparing theresults of replicate assays performed by two different analysts on twodifferent days, or by comparing the results of independent preparationsof different lots of peanut flour on the same or different days.

Precision may be estimated by performing the extraction of a singlesample in triplicate, and analyzing the results according to theproposed method (see, e.g., Table 1). Triplicate extractions anddeterminations of a single lot of peanut flour are conducted; reportedvalues are percent area of each Ara h species. Integration of the peaksmay be performed by using forced integration events on a data system(e.g., ChemStation), or manual integration. The precision for thesetriplicate independent preparations of a single lot of peanut flour mayrange from about 1.1% relative standard deviation (RSD) for Ara h6 toabout 18.3% for Ara h1. The higher (% RSD) for Ara h1 may be associatedwith integrating the Ara h1 shoulder from the subsequent larger cluster.

TABLE 1 RP-HPLC Method Precision % Area Peanut Ara h1 Flour Lot # Ara h2Ara h6 (shoulder) 112FA02411 12.20 6.36 7.41 11.95 6.30 9.68 12.30 6.2210.72 Average 12.15 6.30 9.27 Std Dev 0.1762 0.0718 1.6955 % RSD 1.45%1.14% 18.29%

A second precision method compares the results obtained by two differentanalysts performing the assay on two different days. Each valuepresented represents the average of duplicate injections. Table 2provides exemplary results of a comparison of the percent area valuesand extractable protein content of three peanut flour lots, by twodifferent analysts on different days. Comparison of the quantitativeresults obtained from these assays yields Ara h values that agreebetween 86% to 107%; total protein content may agree within 95%-102%.The percent of the match between the two analysts may also be presented.

TABLE 2 RP-HPLC Method Precision % Area Ara h2 Ara h6 Ara h1 (shoulder)Peanut Ana- Ana- Ana- Ana- Ana- Ana- Flour Lot # lyst 1 lyst 2 lyst 1lyst 2 lyst 1 lyst 2 111FA36111 10.60 12.29 5.59 5.77 8.82 9.73 % Match86.31 96.76  90.70 111FA36211 10.65 12.02 5.48 5.67 11.14 9.36 % Match88.58 96.63 118.97 112FA02411 10.62 12.15 5.93 6.30 9.91 9.27 % Match87.40 94.12 106.92 Values are the average of two injections

Analysis of various PF lots may be used to demonstrate that theexpression of Ara h1, Ara h2 and Ara h6 is consistent, both individuallyand relative to each other across lots of peanut flour. This assay mayalso form the basis for identity and content testing at release andduring stability determination.

The assay was be conducted and analyzed by a second cGMP manufacturer.The HPLC profiles (see, e.g., FIG. 2, FIG. 3 and FIG. 4), total proteinand percentage of each allergen within the total protein (see, e.g.,Table 3) are generally consistent between the assays performed by bothlaboratories using the same peanut flour lots (allows bridging of thedata).

TABLE 3 Comparison for Ara h Proteins and Total Extractable ProteinContent % Area Ara h2 Ara h6 Ara h1 % Peanut Flour Lot (shoulder)(shoulder) (shoulder) Protein 111FA36111 11.40 5.29 9.79 11.26111FA36211 11.20 5.67 9.85 11.03 112FA02411 (Non GMP) 11.31 5.68 10.4110.23 112FA02411 (GMP) 10.58 5.79 10.16 10.25

RP-HPLC Confirmation Studies

To confirm that the RP-HPLC peak profile actually separates andidentifies Ara h1, Ara h2 and Ara h6, material isolated from each peakmay be further characterized by SDS polyacrylamide gel electrophoresisusing, for example, a 4-20 Novex Tris-HCl pre-cast gel (see, e.g., FIG.5). Additional gels may be transferred to polyvinylidene difluoride(PVDF) membranes, processed for immunoblotting and may be reacted withAra h1, Ara h2 or Ara h6 chicken antisera and developed with horseradish peroxidase conjugated goat anti-chicken IgG using, for example,an assay method described by de Jong et al. (EMBO J., 1988; 7(3):745-750). It should be noted that while extracts may be derived fromroasted peanut flour, the antisera may be generated against Ara hproteins purified from raw peanut extracts. The antisera react with boththe control Ara h proteins derived from raw peanuts and from theisolated Ara h proteins obtained from roasted peanut extracts (see,e.g., FIG. 6).

The immunoblots show that the material isolated from each of the threeprinciple HPLC peaks was reactive with the appropriate peanut proteinspecific antisera, and that the molecular weight of the immunoreactiveproteins corresponded to the protein molecular weights as reported inthe literature (Koppelman et al. 2010). It was determined that the Ara hproteins extracted from the peanut flour are not sensitive to heating to60° C. Additional confirmatory experiments may be conducted; theseassays may be used to establish the most appropriate stabilityindicating assay that provides the greatest sensitivity to changesoccurring during long-term storage. However, the early immunoblot datadescribed herein indicate that the reported RP-HPLC method will trackthe individual peanut proteins among peanut flour lots.

Source and Testing of the Peanut Flour

Peanut Flour (PF) for use in a formulation described herein may besourced from any reliable producer including, but not limited to, theGolden Peanut Company (GPC) which manufactures peanut flour and peanutoil (a byproduct of defatting the roasted peanuts).

A GPC manufacturing facility may be audited by an internationallyrecognized certification body for food safety programs (e.g., IntertekLabtest (UK) Limited). The audit may focus on compliance with theBritish Retail Consortium Food Standard (BRC) Global Standards for FoodSafety. The BRC Global Standards are a leading global safety and qualitycertification program, used throughout the world by over 17,000certificated suppliers in 90 countries through a network of over 80accredited and BRC recognized Certification Bodies. The BRC GlobalStandards are widely used by suppliers and global retailers. Theyfacilitate standardization of quality, safety, operational criteria andmanufacturers' fulfillment of legal obligations. They also help provideprotection to the consumer. There were no major or criticalnon-conformity findings during the most recent audit.

The PF may be about 12% defatted peanut flour milled from lightlyroasted peanuts. The PF may be by the supplier after standard analysisof content and microbiology, and is identified as stable for 9 monthsunder refrigeration.

Incoming Raw Material Release Testing for PF

The PF raw material may be tested for appearance, identify, totalprotein content and moisture content prior to release for cGMPproduction (see, e.g., Table 4). The PF may be stored under controlledconditions at 2-8° C.

TABLE 4 Raw Material Testing for PF Assay Method Acceptance CriteriaAppearance Visual Fine powder Powder/Color Tan color Identity RP-HPLCComparable to Reference Chromatogram Protein Content Nitrogen Content byAOCS Report Results Combustion Method for Determination of Crude Protein(AOCS Official Method Ba 4e−93) Moisture Loss on Drying (LOD) ReportResults USP <921>

Formulation Excipients

Table 5 provides exemplary excipients that may be used in a formulationdescribed herein. Other excipients that may be used in a formulationdescribed herein are provided elsewhere in the description.

Exemplary intended dosage form include, for example, a HydroxypropylMethyl Cellulose (HPMC) based capsule; the strength of the dosage formmay be about 0.5 mg, about 1 mg. about 10 mg, about 100 mg, about 475mg, or about 1000 mg of peanut protein. The peanut protein itself, insome instances, may be a cohesive material without inherent flowproperties conducive to conventional pharmaceutical manufacturingprocesses. Thus, inactive pharmaceutical ingredients (excipients) may beadded to the formulation so the peanut flower may be developed into aproper pharmaceutical dosage form with flow characteristics to enhanceboth manufacturing and also delivery of the dosage form.

Compatibility studies may be conducted to evaluate combinations ofpeanut flour with exemplary excipient categories (diluent, glidant andlubricant). The excipients may have GRAS recognition or be shown to besafe in pharmaceutical formulations. The diluent provides theopportunity to formulate the low and high doses to contain adequatevolume for dispersal from the opened capsule. The glidant and lubricantadd flowability to the PF such that the capsule is easily emptied offlour by the subject.

As per Table 5 each of the excipients under consideration are designatedas USP, NF or USP-NF.

TABLE 5 Excipients Under Consideration Manufacturer FunctionalityExcipient (Trade Name) Grade Description Diluents Lactose MonohydrateForemost NF Simple Organic Diluent Lactose (Monohydrate) 316/Fast-Flo)Lactose Kerry/Sheffield NF Simple Organic Diluent Anhydrous (Lactose DT)(Anhydrous) Mannitol Roquette NF Simple Organic Diluent (Pearlitol300DC) Microcrystalline FMC NF Complex Organic Cellulose (Avicel pH102)Diluent Partially Colorcon USP/NF Complex Organic Pregelatinized (Starch1500) Diluent Corn Starch Silicified JRS Pharma USP ComplexMicrocrystalline (PROSOLV Organic/Inorganic Co- Cellulose HD90)processed Diluent Dicalcium Phosphate Innophos NF Inorganic Diluent(DiTab) Glidant Colloidal Silicon Cabot USP Glidant/Anticaking Dioxide(Cab-O-Sil Agent M5P) Talc Ultra Chemicals USP Glidant/Anticaking (UltraTalc Agent 4000) Lubricants Magnesium Stearate Mallinckrodt USPLubricant (vegetable source) Sodium Stearyl JRS Pharma USP LubricantFumarate (Pruv) Capsule Shell White Opaque HPMC Capsugel n/a VegetableSource Capsule Shell (V-Caps) Capsule Shell Capsule Pigment BlendsV54.9041 TBD Representative of final Coloring V18.9221 capsule shellcolor Agents V41.9071 Caramel Color Sensient TBD Colorant for matchingplacebo blends

Formulation of the Characterized Peanut Allergen

Peanut flour (containing peanut allergen proteins Ara h1, Ara h2 and Arah6) may be formulated with a bulking and a flow agent in graduateddoses, comprising capsules containing 0.5 mg, 1 mg, 10 mg, 100 mg and1000 mg each of peanut protein.

Low Dose Capsules (0.5 mg and 1 mg)

FIG. 7 and Table 6 outline the proposed blending process for the lowdose capsules, which include the 0.5 mg peanut protein and 1 mg peanutprotein capsules.

Provided herein is a method of making a low dose capsule formulationuseful in the methods provided here, comprising, (a) mixing peanut flourand diluent in a first blend; (b) adding about 45% of diluent in asecond blend; (c) adding remaining diluent and/or lubricant in a thirdblend; (d) adding a glidant in a final blend; and (e) encapsulatingblended powder in a capsule. In one embodiment, the diluent of step (a)comprises starch, lactose or microcrystalline cellulose (Avicel®), ordicalcium phosphate. In another embodiment, the diluent of step (b)and/or (c) comprises starch, lactose or microcrystalline cellulose(Avicel®), or dicalcium phosphate. In another embodiment, the glidant ofstep (d) glidant of step (d) comprises colloidal silicon dioxide(Cab-O-Sil), talc (e.g., Ultra Talc 4000), or combinations thereof. Inanother embodiment, the glidant of step (d) comprises Cab-O-Sil. Inanother embodiment, the lubricant of step (d) comprises magnesiumstearate. In another embodiment, the method further comprises samplingthe blended mixture one or more times prior to encapsulation. In anotherembodiment, the dose comprises about 0.5 or about 1.0 mg peanut protein.In one embodiment, the method optionally comprises sampling the blendedmaterial of step (d). In one embodiment, step (d) comprises adding aglidant or a lubricant. In another embodiment, step (d) comprises addinga glidant and a lubricant.

TABLE 6 Proposed Operation Steps for Low Dose Capsules (0.5 mg and 1 mg)Operation Equipment Step Type Details Comments 1 Diffusion V-BlenderBlender shell size TBD based on batch size Blender Use of intensifierbar dependent on uniformity results from developmental batches 2Diffusion V-Blender Blender shell size TBD based on batch size Blender 3Diffusion V-Blender Blender shell size TBD based on batch size Blender 4Diffusion V-Blender Blender shell size TBD based on batch size Blender 5Sample Thief TBD Thief length and chamber size appropriate for blendersize and analytical sample size requirements 6 Encapsulator Dosing Disk/Encapsulation method TBD based on fill Auger weight variationassessments in developmental batches

High Dose Capsules (10 mg, 100 mg and 475 mg)

FIG. 8 and Table 7 outline the proposed blending process for the highdose capsules, which include the 10 mg peanut protein, 100 mg peanutprotein and 475 mg peanut protein capsules.

Provided herein is a method of making a high dose capsule formulationuseful in the methods provided here, comprising, (a) mixing peanut flourand diluent in a first blend; (b) discharging the blended material; (c)passing the blended material through a mesh screen and blending thescreened material in a second blend; (d) adding in a glidant and/orlubricant in a third blend; (e) encapsulating the blended powder. In oneembodiment, the method optionally comprises sampling the blendedmaterial of step (d) one or more times prior to encapsulation. In yetanother embodiment, the diluent of step (a) comprises starch, lactose ormicrocrystalline cellulose (Avicel®), or dicalcium phosphate. In anotherembodiment, the mesh screen of step (c) comprises a #20 mesh screen. Inanother embodiment, the glidant of step (d) glidant of step (d)comprises colloidal silicon dioxide (Cab-O-Sil), talc (e.g., Ultra Talc4000), or combinations thereof. In another embodiment, the glidant ofstep (d) comprises Cab-O-Sil. In another embodiment, the lubricant ofstep (d) comprises magnesium stearate. In one embodiment, step (d)comprises adding a glidant or a lubricant. In another embodiment, step(d) comprises adding a glidant and a lubricant.

TABLE 7 Proposed Operation Steps for High Dose Capsules (10 mg, 100 mgand 475 mg) Operation Equipment Step Type Details Comments 1 DiffusionV-Blender Blender shell size TBD based Blender on batch size 2 Sieve U.SSTD Promote blend uniformity #20 (850 μm) 3 Diffusion V-Blender Blendershell size TBD based Blender on batch size 4 Diffusion V-Blender Blendershell size TBD based Blender on batch size 5 Sample TBD Thief length andchamber size Thief appropriate for blender size and analytical samplesize requirements 6 Encapsulator Dosing Disk/ Encapsulation method TBDAuger based on fill weight variation assessments in developmentalbatches

Control of the Bulk Substance

Exemplary proposed specifications for formulated Bulk Substance aresummarized in Table 8.

TABLE 8 Proposed Specifications for Bulk Substance Attribute MethodAcceptance Criteria General Appearance Visual TBD Powder/Color MoistureLOD Report Results Identity Presence of Ara h1, Reverse Phase HPLCComparable to Reference Ara h2 and Ara h6 Chromatogram Report proteinspercent area of Ara h1, Ara h2 and Ara h6 Strength Total ProteinNitrogen Content by Low doses (0.5 and 1 mg): (Assay) Determination AOCSTarget protein concentration Combustion Method for ±15% Determination ofCrude High doses (10, 100 and Protein 475 mg): Target protein (AOCSOfficial Method concentration Ba 4e−93) ±10% Safety BioburdenMicrobiological Limits Total Aerobic Microbial USP <61> Count: MicrobialEnumeration NMT 1000 CFU/g USP <62> Total Yeasts & Molds SpecifiedCount: Microorganisms NMT 100 CFU/g E. coli: Absent S. aureus: Absent P.aeruginosa: Absent Salmonella species: Absent

Bulk Stability Testing

The formulation may be filled into capsules within 24 hours of blending.

Formulation

Overview of Chemistry and Manufacturing Composition

Peanut flour (containing peanut allergen proteins Ara h1, Ara h2 and Arah6) may be formulated with a bulking and a flow agent in graduateddoses, comprising capsules comprising about 0.5 mg, about 1 mg, about 10mg, about 100 mg, about 475 mg, or about 1000 mg each of peanut proteinwith one or more diluents, one or more glidants, one or more lubricants.Optionally one or more filling agents may be added. Each capsule may beopened and the content mixed into taste-masking food immediately priorto administration.

Non-animal capsules that meet global Pharmaceutical standards may beused for the formulations described herein. In one non-limitingembodiment, HPMC capsules from Capsugel may be used.

In another non-limiting embodiment, capsules may be color coded todistinguish the different doses Matching color-coded placebo capsulesmay also be produced.

TABLE 9 Exemplary Dosage Forms Peanut Protein Dose Capsule Size 1  0.5mg 3 2  1 mg 3 3  10 mg 00 4 100 mg 00 5 475 mg 000

The final excipient composition of the formulation may be determinedafter completion of the ongoing compatibility study with the differentexcipients (see Table 5).

Manufacturing Process

Encapsulation method/equipment may be determined based on fill weightvariation assessments in developmental batches. In-process controls mayinclude periodic weight checks.

Control of the Formulation

Exemplary release specifications of the formulations are presented inTable 10.

TABLE 10 Proposed Specifications for the Formulation Attribute MethodAcceptance Criteria General Appearance Visual TBD Powder/color CapsuleIntegrity Visual Intact capsules with no visible signs of cracking.Capsules open easily without breaking Content Uniformity USP <905> MeetsUSP <905> requirements Deliverable Mass % Weight Report resultsDelivered Moisture Loss on Drying (LOD) Report Results USP <921>Identity Presence of Ara h1, Reverse Phase HPLC Comparable to referenceAra h2 and Ara h6 chromatogram proteins and Report percent area of Arah1, Ara h2 and Ara h6 Strength Protein Content Nitrogen Content by Lowdoses (0.5 and 1 mg): (Assay) AOCS Combustion Target protein Method forconcentration ±15% Determination of Crude High doses (10 and Protein 100mg): (AOCS Official Method Target protein Ba 4e−93) concentration ±10%Safety Bioburden Microbiological Limits Total Aerobic Microbial USP <61>Count: NMT 1000 CFU/g Microbial Total Yeasts & Molds Enumeration Count:USP <62> NMT 100 CFU/g Specified E. coli: Absent Microorganisms S.aureus: Absent P. aeruginosa: Absent Salmonella species: Absent

Appearance

Appearance assessments may be performed on the bulk substance (e.g.,formulation during one or more preparation steps and/or of the finalmixture prior to encapsulation) and the formulation. Assessment of theappearance may include, for example, consists of visually inspecting thecontainer against a white background illuminated by a full spectrumlight.

Content Uniformity

Content uniformity (CU) of capsules may be performed according to USPstandards. Content uniformity may be based on a total protein nitrogencontent combustion assay. The intent is to identify a combustioninstrument with the sensitivity to enable assaying individual capsulesat all doses.

Deliverable Mass

The capsule deliverable mass may be evaluated by weighing capsules, andemptying the contents, and weighing the emptied capsules. The %delivered amount may then be calculated.

Moisture Content

Moisture content may impact the stability of proteins, and understandingthe changes in moisture content over time is useful for understandingchanges in the formulation that may, in some instances, lead to shortershelf life. For peanut flour filled capsules, moisture content may bemeasured using Loss on Drying (LOD) determinations according to the USP.Conditions for the LOD may be determined based on the excipientsrequirements and requirements for the peanut flour.

Identity (RP-HPLC)

RP-HPLC may be used to confirm identity of the PF, BS and finalformulation. Samples may be analyzed according to the methods describedin more detail in the related application entitled “Peanut Formulationsand Uses Thereof”, filed the same day herewith (Attorney Docket No.43567-702.101), which is incorporated herein by reference in itsentirety, and the resulting chromatograms may be compared to the examplechromatogram provided in the test method (See, e.g., FIG. 9).

A positive identification of peanut flour may be confirmed if the samplechromatogram matches the chromatogram provided in the method. If apositive indication is not confirmed, a lot of peanut flour may bediscarded as sub-standard. Absence of active in placebos may beconfirmed by demonstrating that no peaks elute between 12 and 35 minutesin the chromatography.

Total Extractable Protein

A similar approach to the determination of total extractable protein inpeanut flour may be used for the determination of total extractableprotein in the capsule formulations. The approach may be evaluated forall strengths. In brief, capsule contents may be emptied, weighed, andanalyzed by RP-HPLC. Chromatographic analysis of peanut flour samplesextracted using this procedure produce a chromatographic “fingerprint”that is unique to peanut flour extracts. The region of the samples thatelute between approximately 12 minutes and 35 minutes may be integrated.The total area integrated may be quantitated against a BSA standard. Thetotal extractable protein content may then calculated using thefollowing equation.

${{Mg}\text{/}g\mspace{14mu} {protein}} = {\frac{R_{u}}{R_{s}} \times C_{STD} \times \frac{V_{Sample}}{{Wt}_{Sample}}}$

where:

-   -   R_(u)=Total Ara h Protein Peak Area or Ara h Species Peak Area        in the Working Sample;    -   R_(s)=Average BSA Peak Area in all Working Standards CSTD=BSA        Working Standard Concentration (mg/mL);    -   V_(Sample)=Total Diluent Volume of the Working Sample (10.0 mL);        and    -   Wt_(Sample)=Weight of peanut flour sample (g).

Apparent Ara h1, Ara h2 and Ara h6 Protein Ratios

Chromatographic analysis of samples extracted using the RP-HPLC methodmay produce a chromatographic “fingerprint” that is unique to peanutflour extracts, and relative ratios of regions corresponding to Ara h1,Ara h2, and Ara h6 (see, e.g., FIG. 1). The protein content of each ofthese regions (mg/g) may be quantitated according to the equationprovided above. Relative percent content of total protein for eachregion is then calculated according to the equation below.

${{Ara}\mspace{14mu} h\mspace{14mu} \%} = {\frac{{Ara}\mspace{14mu} h\mspace{14mu} {PeakArea}}{{{Total}/{protein}}\mspace{14mu} {PeakArea}} \times 100}$

Protein Content

Protein content in filled capsules may be determined in the same manneras that of the peanut flour (AOCS Official Method Ba 4e-93). Since theaccurate protein content determinations may be dependent on the nitrogencontent of the sample, no excipients containing nitrogen may be used inthe formulation. The method is based on the Dumas method and is based onthe combustion of the crude protein in pure oxygen, and measurement ofthe nitrogen gas that is evolved. The method that may be used may beAOCS Official Method Ba 4e-93. The AOCS Method Definition and Scope areprovided below.

Briefly, this method describes a generic combustion method for thedetermination of crude protein. Combustion at high temperature in pureoxygen frees nitrogen, which is measured by thermal conductivitydetection and then converted to equivalent protein by an appropriatenumerical factor. This is an alternative method to the mercury catalystKjeldahl method and has two advantages: 1) less time is needed fornitrogen determination, and 2) hazardous and toxic chemicals are notutilized.

Stability Testing

Formulations may be stored at 2-8° C. To assess accelerated andlong-term stability, formulations may be tested according to thefrequency and specifications described in Table 11 and Table 12. Testingfor appearance/color, moisture, identity and strength may be performedat all timepoints, and the bioburden may be performed annually at 12,24, and 36 months.

TABLE 11A Stability Protocol Testing Scheme for a FormulationTemperature 25° C. +/− 2° C. 5° C. +/− 3° C. 60% RH Testing 1, 3, 6, 9,12, 18, 24, 36 1, 3, 6, 9, 12, 18, 24, 36 Frequency months months

Tables 11B-11F provide data obtained by testing stability of variousformulations at 5° C.

TABLE 11B Stability Condition: 5° C. Characterized Peanut Allergen, 475mg Capsule Specifications Stability Intervals Acceptance 1 3 6 TestMethod Criteria Initial Month Month Month Identi- TM-074 Report Area10.18 8.5 9.67 9.31 fication % Ara h1 (HPLC) Report Area 9.48 9.89 10.888.93 % Ara h2 Report Area 5.89 5.16 5.32 4.21 % Ara h6 Report the 1.611.92 2.05 2.12 ratio of Ara h2/h6

TABLE 11C Stability Condition: 5° C. Characterized Peanut Allergen, 100mg Capsule Specifications Stability Intervals Acceptance 1 3 6 TestMethod Criteria Initial Month Month Month Identi- TM-074 Report Area7.97 10.33 10.51 9.64 fication % Ara h1 (HPLC) Report Area 8.81 8.789.01 8 % Ara h2 Report Area 4.17 3.92 4.27 3.61 % Ara h6 Report the 2.112.24 2.11 2.22 ratio of Ara h2/h6

TABLE 11D Stability Condition: 5° C. Characterized Peanut Allergen, 10mg Capsule Specifications Stability Intervals Acceptance 1 3 6 TestMethod Criteria Initial Month Month Month Identi- TM-074 Report Area6.66 7.71 9.36 7.11 fication % Ara h1 (HPLC) Report Area 10.95 9.75 9.5410.16 % Ara h2 Report Area 5.93 5.8 5.55 5.51 % Ara h6 Report the 1.851.68 1.72 1.84 ratio of Ara h2/h6

TABLE 11E Stability Condition: 5° C. Characterized Peanut Allergen, 1.0mg Capsule Specifications Stability Intervals Acceptance 1 3 6 TestMethod Criteria Initial Month Month Month Identi- TM-074 Report Area7.35 7.43 8.54 7.65 fication % Ara h1 (HPLC) Report Area 16.11 14.3912.31 12.94 % Ara h2 Report Area 7.14 6.56 5.77 6.36 % Ara h6 Report the2.26 2.19 2.13 2.03 ratio of Ara h2/h6

TABLE 11F Stability Condition: 5° C. Characterized Peanut Allergen, 0.5mg Capsule Specifications Accep- Stability Intervals tance Ini- 1 3 6 9Test Method Criteria tial Month Month Month Month Identi- TM-074 Report7.12 8.25 8.3 8 6.09 fication Area (HPLC) % Ara h1 Report 19.37 14.7615.26 16.15 20.78 Area % Ara h2 Report 8.77 8.69 8.9 8.8 10.38 Area %Ara h6 Report 2.21 1.7 1.71 1.84 2.00 the ratio of Ara h2/h6

Tables 11G-11K provide data obtained by testing stability of variousformulations at 25° C.

TABLE 11G Stability Condition: 25° C. Characterized Peanut Allergen, 475mg Capsule Specifications Stability Intervals Acceptance 1 3 6 TestMethod Criteria Initial Month Month Month Identi- TM-074 Report Area10.18 8.26 10.1 9.93 fication % Ara h1 (HPLC) Report Area 9.48 9.8610.48 9.77 % Ara h2 Report Area 5.89 5.09 5.25 4.41 % Ara h6 Report the1.61 1.94 2 2.22 ratio of Ara h2/h6

TABLE 11H Stability Condition: 25° C. Characterized Peanut Allergen, 100mg Capsule Specifications Stability Intervals Acceptance 1 3 6 TestMethod Criteria Initial Month Month Month Identification TM-074 ReportArea 7.97 9.92 10.42 9.75 (HPLC) % Ara h1 Report Area 8.81 8.32 9.4 8.04% Ara h2 Report Area 4.17 4.18 4.28 3.6 % Ara h6 Report the 2.11 1.992.2 2.23 ratio of Ara h2/h6

TABLE 11I Stability Condition: 25° C. Characterized Peanut Allergen, 10mg Capsule Specifications Stability Intervals Acceptance 1 3 6 TestMethod Criteria Initial Month Month Month Identification TM-074 ReportArea 6.66 7.99 9.47 7.26 (HPLC) % Ara h1 Report Area 10.95 10.77 10.2310.11 % Ara h2 Report Area 5.93 5.81 4.99 5.83 % Ara h6 Report the 1.851.85 2.05 1.73 ratio of Ara h2/h6

TABLE 11J Stability Condition: 25° C. Characterized Peanut Allergen, 1.0mg Capsule Specifications Stability Intervals Acceptance 1 3 6 TestMethod Criteria Initial Month Month Month Identification TM-074 ReportArea 7.35 7.63 8.24 7.74 (HPLC) % Ara h1 Report Area 16.11 12.59 12.9712.89 % Ara h2 Report Area 7.14 6.55 5.81 6.05 % Ara h6 Report the 2.261.92 2.23 2.13 ratio of Ara h2/h6

TABLE 11K Stability Condition: 25° C. Characterized Peanut Allergen, 0.5mg Capsule Specifications Stability Intervals Acceptance 1 6 Test MethodCriteria Initial Month 3 Month Month 9 Month Identification TM-074Report Area 7.12 8.22 7.95 7.83 6.16 (HPLC) % Ara h1 Report Area 19.379.49 16.3 16.28 20.92 % Ara h2 Report Area 8.77 15 8.76 8.2 9.47 % Arah6 Report the 2.21 1.58 1.86 1.99 2.21 ratio of Ara h2/h6

TABLE 12A Stability Protocol Specifications for a Formulation AttributeMethod Acceptance Criteria General Appearance Visual TBD Powder/colorCapsule Integrity Visual Intact capsules with no visible signs ofcracking. Capsules open easily without breaking Moisture Loss on Drying(LOD) Report Results USP <921> Identity Presence of Ara h1, ReversePhase HPLC Comparable to reference Ara h2 and Ara h6 chromatogram andproteins report percent area of Ara h1, Ara h2 and Ara h6 StrengthProtein Content Nitrogen Content by Low doses (0.5 and 1 mg): (Assay)AOCS Combustion Target protein Method for concentration ±15%Determination of Crude High doses (10 and 100 mg): Protein Targetprotein (AOCS Official Method concentration ±10% Ba 4e−93) SafetyBioburden* Microbiological Limits Total Aerobic Microbial USP <61>Count: NMT 1000 CFU/g Microbial Enumeration Total Yeasts & Molds USP<62> Count: Specified NMT 100 CFU/g Microorganisms E. coli: Absent S.aureus: Absent P. aeruginosa: Absent Salmonella species: Absent*Bioburden may be measured at release and annually.

Tables 12B-12K provide data obtained by assessing stability andcharacteristics of various formulations at 5° C. and 25° C. at varioustime points.

TABLE 12B Stability Conditions: 5° C.; 0.5 mg capsule SpecificationsAcceptance Stability Intervals Test Method Criteria Initial 1 Mo 3 Mo 6Mo 9 Mo Appearance Visual White opaque Conforms Conforms ConformsConforms Conforms (n = 10) capsule containing white to off-white finegranular powder* Deliverable TM-086 >95%* Average: Average: Average:Average: Average: Mass 99%; 99%; 100%; 99%; 99%; RSD: RSD: RSD: RSD:RSD: 0.4% 0.5% 0.3% 0.1% 0.6% Assay TM-085 Target protein   91%   88%  92%  101%   88% concentration ±15% Identification TM-074 Comparable toComparable Comparable Comparable Comparable Comparable (HPLC) referencechromatogram Report Area % 7.12 8.25 8.3 8 6.09 Ara h1 Report Area %19.37 14.76 15.26 16.15 20.78 Ara h2 Report Area % 8.77 8.69 8.9 8.810.38 Ara h6 Report the ratio of 2.21 1.7 1.71 1.84 2.00 Ara h2/h6 Losson USP Report Results 3.83% 4.00% 4.50% 6.40% 5.40% Drying <731> (@130°C. for 2 hours) Microbial USP <61> Total Aerobic Meets NA NA NA NALimits/ and <62> Microbial Count: Acceptance Specified Quality NMT 1000cfu/g; Criteria Microorganisms Chemical Total Combined LaboratoriesYeasts & Molds Count: NMT 100 cfu/g; E. coli, S. aureus, P. aeruginosaand Salmonella species are absent

TABLE 12C Stability Condition: 25° C./60% RH; 0.5 mg capsuleSpecifications Acceptance Stability Intervals Test Method CriteriaInitial 1 Mo 3 Mo 6 Mo 9 Mo Appearance Visual White opaque ConformsConforms Conforms Conforms Conforms (n = 10) capsule containing white tooff-white fine granular powder* Deliverable TM-086 >95%* Average:Average: Average: Average: Average: Mass 99% 100% 99% 100% 99% RSD: RSD:RSD: RSD: RSD: 0.4% 0.5% 0.4% 0.3% 0.7% Assay TM-085 Target protein  91%   90%   90%   98%   82% concentration ±15%; (85-115% label claim)Identification TM-074 Comparable to Comparable Comparable ComparableComparable Comparable (HPLC) reference chromatogram Report Area % 7.128.22 7.95 7.83 6.16 Ara h1 Report Area % 19.37 9.49 16.3 16.28 20.92 Arah2 Report Area % 8.77 15 8.76 8.2 9.47 Ara h6 Report the ratio of 2.211.58 1.86 1.99 2.21 Ara h2/h6 Loss on USP Report Results 3.83% 3.70%4.20% 4.10% 4.60% Drying <731> (@130° C. for 2 hours) Microbial USP <61>Total Aerobic Meets NA NA NA NA Limits/ and <62> Microbial Count:Acceptance Specified Quality NMT 1000 cfu/g; Criteria MicroorganismsChemical Total Combined Laboratories Yeasts & Molds Count: NMT 100cfu/g; E. coli, S. aureus, P. aeruginosa and Salmonella species areabsent

TABLE 12CD Stability Condition: 5° C.: Characterized Peanut Allergen,1.0 mg Capsule Specifications Acceptance Stability Intervals Test MethodCriteria Initial 1 Mo 3 Mo 6 Mo Appearance Visual White ConformsConforms Conforms Conforms (n = 10) opaque capsule containing white tooff- white fine granular powder* Deliverable TM-086 >95%* Average:Average: Average: Average: Mass 100%; 100%; 99%; RSD: 99%; RSD: RSD:RSD: 0.6% 0.3% 0.3% 0.4% Assay TM-085 Target  101%   90%   86%   94%protein concentration ±15%; (85-115% label claim) Identification TM-074Comparable Comparable Comparable Comparable Comparable (HPLC) toreference chromatogram Report Area 7.35 7.43 8.54 7.65 % Ara h1 ReportArea 16.11 14.39 12.31 12.94 % Ara h2 Report Area 7.14 6.56 5.77 6.36 %Ara h6 Report the 2.26 2.19 2.13 2.03 ratio of Ara h2/h6 Loss on USP<731> Report 5.02% 5.20% 5.70% 6.20% Drying Results (@130° C. for 2hours) Microbial USP <61> Total Meets NA NA NA Limits/ and <62> AerobicAcceptance Specified Quality Microbial Criteria Microorganisms ChemicalCount: NMT Laboratories 1000 cfu/g; Total Combined Yeasts & Molds Count:NMT 100 cfu/g; E. coli, S. aureus, P. aeruginosa and Salmonella speciesare absent

TABLE 12E Stability Condition: 25° C./60% RH; Characterized PeanutAllergen, 1.0 mg Capsule Specifications Acceptance Stability IntervalsTest Method Criteria Initial 1 Mo 3 Mo 6 Mo Appearance Visual WhiteConforms Conforms Conforms Conforms (n = 10) opaque capsule containingwhite to off- white fine granular powder* Deliverable TM-086 >95%*Average: Average: Average: Average: Mass 100%; 100%; 99%; RSD: 100%;RSD: RSD: 0.2% RSD: 0.3% 0.3% 0.3% Assay TM-085 Target  101%   90%   87%  94% protein concentration ±15%; (85-115% label claim) IdentificationTM-074 Comparable Comparable Comparable Comparable Comparable (HPLC) toreference chromatogram Report Area 7.35 7.63 8.24 7.74 % Ara h1 ReportArea 16.11 12.59 12.97 12.89 % Ara h2 Report Area 7.14 6.55 5.81 6.05 %Ara h6 Report the 2.26 1.92 2.23 2.13 ratio of Ara h2/h6 Loss on USP<731> Report 5.02% 5.00% 5.80% 6.10% Drying Results (@130° C. for 2hours) Microbial USP <61> Total Meets NA NA NA Limits/ and <62> AerobicAcceptance Specified Quality Microbial Criteria Microorganisms ChemicalCount: NMT Laboratories 1000 cfu/g; Total Combined Yeasts & Molds Count:NMT 100 cfu/g; E. coli, S. aureus, P. aeruginosa and Salmonella speciesare absent

TABLE 12F Stability Condition: 5° C.; Characterized Peanut Allergen, 10mg Capsule Specifications Acceptance Stability Intervals Test MethodCriteria Initial 1 Mo 3 Mo 6 Mo Appearance Visual White ConformsConforms Conforms Conforms (n = 10) opaque capsule containing white tooff- white fine granular powder* Deliverable TM-086 >95%* Average:Average: Average: Average: Mass 100%; 100%; 100%; 100%; RSD: RSD: RSD:RSD: 0.2% 0.1% 0.1% 0.1% Assay TM-085 Target   95%   93%   96%   98%protein concentration ±10% (90-110% label claim) Identification TM-074Comparable Comparable Comparable Comparable Comparable (HPLC) toreference chromatogram Report Area 6.66 7.71 9.36 7.11 % Ara h1 ReportArea 10.95 9.75 9.54 10.16 % Ara h2 Report Area 5.93 5.8 5.55 5.51 % Arah6 Report the 1.85 1.68 1.72 1.84 ratio of Ara h2/h6 Loss on USP <731>Report 4.90% 5.60% 5.40% 5.50% Drying Results (@130° C. for 2 hours)Microbial USP <61> Total Meets NA NA NA Limits/ and <62> AerobicAcceptance Specified Quality Microbial Criteria Microorganisms ChemicalCount: NMT Laboratories 1000 cfu/g; Total Combined Yeasts & Molds Count:NMT 100 cfu/g; E. coli, S. aureus, P. aeruginosa and Salmonella speciesare absent

TABLE 12G Stability Condition: 25° C./60% RH; Characterized PeanutAllergen, 10 mg Capsule Specifications Acceptance Stability IntervalsTest Method Criteria Initial 1 Mo 3 Mo 6 Mo Appearance Visual WhiteConforms Conforms Conforms Conforms (n = 10) opaque capsule containingwhite to off- white fine granular powder* Deliverable TM-086 >95%*Average: Average: Average: Average: Mass 100%; 100%; 100%; 100%; RSD:RSD: RSD: RSD: 0.2% 0.2% 0.1% 0.1% Assay TM-085 Target   95%   93%   93%  97% protein concentration ±10% (90-110% label claim) IdentificationTM-074 Comparable Comparable Comparable Comparable Comparable (HPLC) toreference chromatogram Report Area 6.66 7.99 9.47 7.26 % Ara h1 ReportArea 10.95 10.77 10.23 10.11 % Ara h2 Report Area 5.93 5.81 4.99 5.83 %Ara h6 Report the 1.85 1.85 2.05 1.73 ratio of Ara h2/h6 Loss on USP<731> Report 4.90% 4.60% 5.20% 5.20% Drying Results (@130° C. for 2hours) Microbial USP <61> Total Meets NA NA NA Limits/ and <62> AerobicAcceptance Specified Quality Microbial Criteria Microorganisms ChemicalCount: NMT Laboratories 1000 cfu/g; Total Combined Yeasts & Molds Count:NMT 100 cfu/g; E. coli, S. aureus, P. aeruginosa and Salmonella speciesare absent

TABLE 12H Stability Condition: 5° C.; Characterized Peanut Allergen, 100mg Capsule Specifications Acceptance Stability Intervals Test MethodCriteria Initial 1 Mo 3 Mo 6 Mo Appearance Visual White ConformsConforms Conforms Conforms (n = 10) opaque capsule containing white tooff- white fine granular powder* Deliverable TM-086 >95%* Average:Average: Average: Average: Mass 100%; 100%; 100%; 100%; RSD: RSD: RSD:RSD: 0.1% 0.2% 0.1% 0.1% Assay TM-085 Target   99%   95%   99%   99%protein concentration ±10% (90-110% label claim) Identification TM-074Comparable Comparable Comparable Comparable Comparable (HPLC) toreference chromatogram Report Area 7.97 10.33 10.51 9.64 % Ara h1 ReportArea 8.81 8.78 9.01 8 % Ara h2 Report Area 4.17 3.92 4.27 3.61 % Ara h6Report the 2.11 2.24 2.11 2.22 ratio of Ara h2/h6 Loss on USP <731>Report 4.02% 3.70% 4.40% 4.40% Drying Results (@130° C. for 2 hours)Microbial USP <61> Total Meets NA NA NA Limits/ and <62> AerobicAcceptance Specified Quality Microbial Criteria Microorganisms ChemicalCount: NMT Laboratories 1000 cfu/g; Total Combined Yeasts & Molds Count:NMT 100 cfu/g; E. coli, S. aureus, P. aeruginosa and Salmonella speciesare absent

TABLE 12I Stability Condition: 25° C.; Characterized Peanut Allergen,100 mg Capsule Specifications Acceptance Stability Intervals Test MethodCriteria Initial 1 Mo 3 Mo 6 Mo Appearance Visual White ConformsConforms Conforms Conforms (n = 10) opaque capsule containing white tooff- white fine granular powder* Deliverable TM-086 >95%* Average:Average: Average: Average: Mass 100%; 100%; 100%; 100%; RSD: RSD: RSD:RSD: 0.1% 0.1% 0.1% 0.4% Assay TM-085 Target   99%   96%   98%   97%protein concentration ±10% (90-110% label claim) Identification TM-074Comparable Comparable Comparable Comparable Comparable (HPLC) toreference chromatogram Report Area 7.97 9.92 10.42 9.75 % Ara h1 ReportArea 8.81 8.32 9.4 8.04 % Ara h2 Report Area 4.17 4.18 4.28 3.6 % Ara h6Report the 2.11 1.99 2.2 2.23 ratio of Ara h2/h6 Loss on USP <731>Report 4.02% 4.00% 4.40% 4.80% Drying Results (@130° C. for 2 hours)Microbial USP <61> Total Meets NA NA NA Limits/ and <62> AerobicAcceptance Specified Quality Microbial Criteria Microorganisms ChemicalCount: NMT Laboratories 1000 cfu/g; Total Combined Yeasts & Molds Count:NMT 100 cfu/g; E. coli, S. aureus, P. aeruginosa and Salmonella speciesare absent

TABLE 12J Stability Condition: 5° C.; Characterized Peanut Allergen, 475mg Capsule Specifications Acceptance Stability Intervals Test MethodCriteria Initial 1 Mo 3 Mo 6 Mo Appearance Visual White ConformsConforms Conforms Conforms (n = 10) opaque capsule containing white tooff- white fine granular powder* Deliverable TM-086 >95%* Average:Average: Average: Average: Mass 100%; 100%; 100%; 100%; RSD: RSD: RSD:RSD: 0.0% 0.1% 0.0% 0.1% Assay TM-085 Target   90%   94%   96%   96%protein concentration ±10% (90-110% label claim) Identification TM-074Comparable Comparable Comparable Comparable Comparable (HPLC) toreference chromatogram Report Area 10.18 8.5 9.67 9.31 % Ara h1 ReportArea 9.48 9.89 10.88 8.93 % Ara h2 Report Area 5.89 5.16 5.32 4.21 % Arah6 Report the 1.61 1.92 2.05 2.12 ratio of Ara h2/h6 Loss on USP <731>Report 4.00% 3.60% 3.70% 3.90% Drying Results (@130° C. for 2 hours)Microbial USP <61> Total Meets NA NA NA Limits/ and <62> AerobicAcceptance Specified Quality Microbial Criteria Microorganisms ChemicalCount: NMT Laboratories 1000 cfu/g; Total Combined Yeasts & Molds Count:NMT 100 cfu/g; E. coli, S. aureus, P. aeruginosa and Salmonella speciesare absent

TABLE 12K Stability Condition: 25° C.; Characterized Peanut Allergen,475 mg Capsule Specifications Acceptance Stability Intervals Test MethodCriteria Initial 1 Mo 3 Mo 6 Mo Appearance Visual White ConformsConforms Conforms Conforms (n = 10) opaque capsule containing white tooff- white fine granular powder* Deliverable TM-086 >95%* Average:Average: Average: Average: Mass 100%; 100%; 100%; 100%; RSD: RSD: RSD:RSD: 0.0% 0.1% 0.1% 0.1% Assay TM-085 Target   90%   95%   96%   95%protein concentration ±10% (90-110% label claim) Identification TM-074Comparable Comparable Comparable Comparable Comparable (HPLC) toreference chromatogram Report Area 10.18 8.26 10.1 9.93 % Ara h1 ReportArea 9.48 9.86 10.48 9.77 % Ara h2 Report Area 5.89 5.09 5.25 4.41 % Arah6 Report the 1.61 1.94 2 2.22 ratio of Ara h2/h6 Loss on USP <731>Report 4.00% 3.80% 4.10% 4.50% Drying Results (@130° C. for 2 hours)Microbial USP <61> Total Meets NA NA NA Limits/ and <62> AerobicAcceptance Specified Quality Microbial Criteria Microorganisms ChemicalCount: NMT Laboratories 1000 cfu/g; Total Combined Yeasts & Molds Count:NMT 100 cfu/g; E. coli, S. aureus, P. aeruginosa and Salmonella speciesare absent

Placebo

Placebo may consist of the defined mixture of excipients without the PF.Placebo may be filled in the same color-coded capsules as the activeformulation.

TABLE 13 Placebo Release Specification Attribute Method AcceptanceCriteria Appearance Visual TBD Powder/color Capsule Integrity VisualIntact capsules with no visible signs of cracking. Capsules open easilywithout breaking Content Uniformity USP <905> Meets USP <905>requirements Deliverable Mass % Weight Report results Delivered MoistureLoss on Drying (LOD) Report Results USP <921> Identity Absence of Arah1, Ara Reverse Phase HPLC No peaks detected in the h2 and Ara h6proteins elution region of PF Strength Protein Content Nitrogen Contentby No protein detected (Assay) AOCS Combustion Method for Determinationof Crude Protein (AOCS Official Method Ba 4e−93) Safety BioburdenMicrobiological Limits Total Aerobic Microbial USP <61> Count: NMT 1000CFU/g Microbial Total Yeasts & Molds Count: Enumeration NMT 100 CFU/gUSP <62> E. coli: Absent Specified S. aureus: Absent Microorganisms P.aeruginosa: Absent Salmonella species: Absent

Methods of Use

The pharmaceutical compositions prepared using the methods describedherein may be used to compare various lots of peanut proteins forconsistency of product.

Peanuts and peanut flour are common foods and additives found in manyfood products. The intended clinical use for Characterized PeanutAllergen (CPA) is found in relatively small quantities (0.5 to 4000mg/dose) compared to quantities contained in food and will be deliveredvia the same route as orally ingested peanut-containing products.

Currently, preclinical studies exploring treatment modalities in foodallergy animal models are limited. The principle model for induction ofpeanut allergy in mice is to expose mice by oral gavage to peanutproteins in the form of peanut butter, ground roasted peanuts, orpurified peanut proteins, in combination with cholera toxin. After 3 to6 weekly exposures the mice are challenged to demonstrate an allergicresponse. Mice may be challenged by intraperitoneal injection withsub-lethal doses of with a formulation described herein and scored forreaction severity. The intent is to demonstrate that the principleelicitors of anaphylaxis are specific Ara h proteins, rather than acombination of all peanut proteins. In an immunotherapy protocol, miceare treated with whole peanut extract, extract depleted of Ara hproteins, or with purified Ara h proteins alone. Upon challenge posttreatment, changes in body temperature, symptom score and mouse mastcell protease-1 release mice may be assessed. Mice that are desensitizedto further challenge may be treated with an entire extract or the Ara hprotein combination.

The cellular requirements underlying peanut induced anaphylaxis may bedetermined explored in wild-type C57BL/6, B-cell deficient,CD40L-deficient, mast cell deficient or FccRI 8-chain-deficient micesensitized to peanut proteins. After intraperitoneal challenge with aformulation described herein, anaphylaxis is assessed by measurement ofantigen-specific immunoglobulins (Igs), overall symptom score, bodytemperature, vascular permeability, mast cell mediator release andanaphylactic reactions. The B-cell, mast cell and CD40L deficient micemay be sensitized to peanut proteins as shown by production of IgE, andTh2-associated cytokines. The FcεRI ε-deficient mice may experienceanaphylaxis albeit somewhat less severe than the wild-type animals.

In a model of esophago-gastro-enteropathy induced by long term feedingof peanuts to sensitized mice described by Mondoulet et al., 2012,epicutaneous immunotherapy with a formulation described herein maylessen the severity of gastro-intestinal lesions. (Mondoulet et al.,2012).

Data obtained from these models, which may demonstrate one or more ofthe hallmarks of human food allergic reactions, and are to be consideredwith respect to variability of human food allergy.

Provided herein is a method of identifying a composition for treatmentfor desensitization of peanut allergy in a subject, comprising: (a)determining the concentrations of Ara h1, Ara h2 and Ara h6 in acomposition of peanut flour by RP-HPLC; (b) comparing the concentrationsto the concentrations of a reference standard; and (c) identifying acomposition for desensitization of peanut allergy in a subject, whereinthe sample contains at least the concentrations of Ara h1, Ara h2 andAra h6 of the reference standard.

The method may, in some instances, further comprise administering acomposition described herein to a subject, wherein the compositioncomprises at least the concentrations of Ara h1, Ara h2 and Ara h6 ofthe reference standard.

The method may be used to compare lots of peanut flour and, in someinstances, exclude peanut flour from use in a composition or methoddescribed herein where the sample does not contain at least thereference standard amount of Ara h1, Ara h2 and Ara h6.

While preferred embodiments have been shown and described herein, itwill be obvious to those skilled in the art that such embodiments areprovided by way of example only. Numerous variations, changes, andsubstitutions may now occur to those skilled in the art withoutdeparting from the embodiments. It should be understood that variousalternatives to the embodiments described herein may be employed inpracticing the embodiments. It is intended that the following claimsdefine the scope of the embodiments and that methods and structureswithin the scope of these claims and their equivalents be coveredthereby.

1-36. (canceled)
 37. A method of making an encapsulated peanut flourformulation, comprising: (a) mixing 12% defatted peanut flour comprisinga characterized concentration of each of Ara h1, Ara h2 and Ara h6, witha diluent, a glidant and/or lubricant to form a blended material; (b)discharging the blended material; (c) passing the blended materialthrough a mesh screen; and (d) encapsulating the blended material,whereby the method provides an encapsulated peanut flour formulationthat is stable for at least about 18 to about 36 months.
 38. The methodof claim 37, wherein the encapsulated peanut flour formulation comprisesabout 10 mg to about 100 mg peanut protein.
 39. The method of claim 37,wherein the concentration of Ara h1, Ara h2 and Ara h6 is characterizedby Reverse Phase-High Performance Liquid Chromatography (RP-HPLC). 40.The method of claim 37, wherein the encapsulated peanut flourformulation is stable for at least about 36 months.
 41. The method ofclaim 37, wherein the encapsulated peanut flour formulation is stable ata temperature from about 2° C. to about 8° C.
 42. The method of claim37, wherein the encapsulated peanut flour formulation is stable at atemperature of about 25° C.
 43. The method of claim 37, wherein theencapsulated peanut flour formulation is stable at a temperature fromabout 20° C. to about 30° C.
 44. The method of claim 37, wherein theencapsulated peanut flour formulation is allergenic to a subjectdiagnosed with a peanut allergy.
 45. A method of making an encapsulatedformulation, comprising: (a) mixing peanut flour, a diluent, a glidantand/or lubricant to form a blended material; (b) discharging the blendedmaterial; (c) passing the blended material through a mesh screen; and(d) encapsulating the blended material, whereby the method provides anencapsulated formulation that comprises peanut flour.
 46. The method ofclaim 45, wherein the peanut flour comprises approximately 50% peanutprotein w/w.
 47. The method of claim 45, wherein the peanut flour is 12%defatted peanut flour.
 48. The method of claim 45, wherein theencapsulated formulation is stable for at least about 18 to about 36months.
 49. The method of claim 45, wherein the encapsulated formulationis stable for at least about 36 months.
 50. The method of claim 45,wherein the peanut flour comprises a characterized concentration of eachof Ara h1, Ara h2 and Ara h6.
 51. The method of claim 50, wherein theconcentration of Ara h1, Ara h2 and Ara h6 is characterized by ReversePhase-High Performance Liquid Chromatography (RP-HPLC).
 52. The methodof claim 45 further comprising characterizing the concentration of oneor more of Ara h1, Ara h2 and Ara h6 in the peanut flour.
 53. The methodof claim 45, wherein the encapsulated formulation comprises about 10 toabout 100 mg peanut protein.
 54. The method of claim 45, wherein theencapsulated formulation comprises about 0.5 to about 1.0 mg peanutprotein.
 55. The method of claim 45, wherein the encapsulatedformulation comprises about 10 mg to about 1000 mg peanut protein. 56.The method of claim 45, wherein the encapsulated formulation comprisesabout 0.5 mg, about 1.0 mg, about 10 mg, about 100 mg, about 475 mg, orabout 1000 mg peanut flour.
 57. The method of claim 45, wherein theencapsulated formulation is allergenic to a subject diagnosed with apeanut allergy.
 58. A method of making a stable, allergenic encapsulatedformulation, comprising: (a) mixing 12% defatted peanut flour comprisinga characterized concentration of each of Ara h1, Ara h2 and Ara h6, witha diluent, a glidant and/or lubricant to form a blended material; (b)discharging the blended material; (c) passing the blended materialthrough a mesh screen; and (d) encapsulating the blended material,whereby the method provides an encapsulated formulation that is stablefor at least about 12 months at a temperature from about 20° C. to about30° C.
 59. The method of claim 58, wherein the encapsulated formulationis stable for at least about 18 months to about 36 months.